Download ASPARAGINASE FROM ASPERGILLUS ORYZAE ENCODED BY

ASPARAGINASE FROM ASPERGILLUS ORYZAE ENCODED BY THE
ASPARAGINASE GENE FROM A. ORYZAE
Chemical and Technical Assessment
Prepared by Zofia Olempska-Beer, Ph.D.
1. Summary
This Chemical and Technical Assessment summarizes data and information on the asparaginase
enzyme preparation submitted to JECFA by Novozymes A/S in a dossier dated November 9,
2006 (Novozymes, 2006). This document also discusses published information relevant to the
production organism, Aspergillus oryzae.
Asparaginase catalyses the hydrolysis of the amino acid asparagine to aspartic acid and ammonia.
The asparaginase enzyme preparation described in the Novozymes’ dossier is intended for use
during food processing to reduce the level of asparagine and thereby to decrease the risk of
acrylamide formation. Acrylamide is formed in food from asparagine and reducing sugars when
food products are baked or fried at temperatures above 120oC.
Asparaginase is manufactured by pure culture fermentation of a genetically modified strain of A.
oryzae, which expresses the asparaginase gene derived from A. oryzae. The A. oryzae production
strain was derived from a nonpathogenic A. oryzae strain IFO 4177, also known as strain A1560,
using recombinant DNA techniques and traditional mutagenesis.
Strain A1560 is capable of producing low levels of toxic secondary metabolites, cyclopiazonic
acid, kojic acid, and 3-β-nitropropionic acid, when cultivated in media conducive to the synthesis
of these compounds. Strain A1560 is also known to contain non-functional genes related to
aflatoxin synthesis. Nevertheless, strain A1560 and its derivatives have been used in the
production of enzyme preparations that were shown to be safe for use in food.
Strain A1560 was subjected to numerous genetic modifications using both classical mutagenesis
and molecular biology to reduce its capability of producing secondary metabolites as well as
extracellular enzymes. As a result of these modifications, a host strain, designated as BECh2,
was developed. The BECh2 strain is devoid of genes involved in the synthesis of aflatoxins and
cyclopiazonic acid due to the deletion of a large DNA segment carrying these genes. The strain is
also impaired in its capability to produce kojic acid and 3-β-nitropropionic acid. When cultivated
in media designed to induce the synthesis of mycotoxins, strain BECh2 produced kojic acid at a
level of approximately 15% of that produced by strain A1560 and essentially no 3-βnitropropionic acid. The BECh2 strain was also modified by inactivation of three endogenous
amylase and two protease genes. The inactivation of these genes prevents the synthesis of
extracellular amylases and proteases that would be secreted to the fermentation medium during
production of asparaginase.
The BECh2 strain was subsequently used as a host for the asparaginase gene. The asparaginase
gene was isolated from strain A1560 and introduced into the BECh2 strain on the expression
vector, which was integrated into genomic DNA of the host strain. All DNA comprising the
expression vector is well-characterized and does not result in the production of harmful
substances. The BECh2 strain was also used as a host to construct a number of enzyme
production strains, including one expressing the phospholipase A1 gene from Fusarium
Asparaginase from A. oryzae encoded by the asparaginase gene from A.oryzae (CTA) 2007 - Page 1(7)
venenatum. The specifications for phospholipase A1 from Fusarium venenatum expressed in
Aspergillus oryzae were established by JECFA at the 65th meeting (JECFA, 2005).
Asparaginase is produced during fermentation of the asparaginase production strain and is
secreted to the fermentation broth. The enzyme is subsequently purified, concentrated, and
formulated with appropriate food-grade substances. To confirm that no kojic and 3-βnitropropionic acids are formed under the fermentation conditions used for asparaginase
production, the asparaginase preparation was tested for the presence of these secondary
metabolites. Neither kojic acid nor 3-β-nitropropionic acid were detected in the enzyme
preparation.
The formulated asparaginase is marketed under the name Acrylaway® L for use as a processing
aid during food manufacture to convert asparagine to aspartic acid and reduce the formation of
acrylamide. Asparaginase will be primarily used during production of foods processed at high
temperatures, such as potato chips, French fries, and dough-based products, for example, bread,
cookies, crackers, or pretzels. The asparaginase enzyme preparation complies with the General
Specifications and Considerations for Enzyme Preparations Used in Food Processing (JECFA,
2006).
2. Description
Light brown liquid.
3. Method of manufacture
3.1. Aspergillus oryzae
A. oryzae is a filamentous fungus that occurs in soil mainly in Japan and China. It has been used
in Asia in the production of fermented foods such as soy sauce, miso and the rice wine sake for
over 2000 years. At the beginning of the 20th century, A. oryzae was used as a source of αamylase, the first enzyme produced on the industrial scale for use in food (Nielsen et al., 1994).
Since then, A. oryzae has been used in the production of numerous native and heterologous
enzymes for use in food processing. A. oryzae is not known to be pathogenic. Some strains of A.
oryzae may produce low levels of secondary metabolites with low to moderate toxicity, 3-βnitropropionic acid, kojic acid, and cyclopiazonic acid (reviewed by Barbesgaard et al., 1992;
Blumenthal, 2004; Olempska-Beer et al., 2006). The synthesis of these secondary metabolites
can be limited or avoided by using appropriate fermentation conditions. According to several
recent reports, certain strains of A. oryzae contain structural and regulatory genes involved in the
synthesis of aflatoxins. Nevertheless, these strains did not produce aflatoxins even under
conditions that favor aflatoxin synthesis (Watson, et al., 1999; van der Broek et al., 2001;
Tominaga et al., 2006).
3.2. Asparaginase production strain
The asparaginase production strain was derived from the well-known A. oryzae industrial strain
IFO 4177 (also known as A1560) originally obtained from the Institute for Fermentation in
Osaka, Japan. Strain A1560 is known to contain genes involved in the synthesis of 3-βnitropropionic acid, kojic acid, and cyclopiazonic acid. These secondary metabolites are
synthesized under nutritionally limiting conditions rather than under conditions that are optimized
Asparaginase from A. oryzae encoded by the asparaginase gene from A.oryzae (CTA) 2007 - Page 2(7)
for industrial production of enzymes. Strain A1560 also contains inactive genes related to
aflatoxin synthesis.
Strain A1560 was genetically modified by site-directed disruption of three endogenous amylase
genes, one alkaline protease gene, and one neutral metalloprotease I gene. The inactivation of
these genes prevents the synthesis of extracellular amylases and proteases that would be secreted
to the fermentation medium during production of asparaginase. The modified strain was
designated as JaL 228.
To reduce the potential for producing secondary metabolites, strain JaL 228 was exposed to γradiation and screened for cyclopiazonic acid production. A mutant deficient in cyclopiazonic
acid synthesis (designated as BECh1) was selected and analyzed using standard genetic methods.
The genetic analysis showed that the segment of one chromosome containing genes involved in
the synthesis of aflatoxins and cyclopiazonic acid was deleted. The deletion of these genes is
permanent and reversion to the aflatoxin or cyclopiazonic acid production capability is not
possible. Therefore, the BECh1 strain and any strains derived thereof will be incapable of
producing aflatoxins and cyclopiazonic acid regardless of the cultivation conditions. To confirm
the results of the genetic analysis, the BECh1 and A1560 strains were assessed under conditions
optimized for production of secondary metabolites. Under these conditions, neither strain
produced aflatoxins and related compounds (sterigmatocystin and 5-methoxy-sterigmatocystin),
as expected. While the A1560 strain produced cyclopiazonic acid and kojic acid, the BECh1
strain produced only kojic acid. Additional growth media were subsequently developed to test
the BECh1 strain for production of 3-β-nitropropionic acid. The compound was produced at a
trace level only under one set of very specific cultivation conditions.
The BECh1 strain was subsequently subjected to UV mutagenesis. A mutant impaired in kojic
acid synthesis was selected and designated as BECh2. The BECh2 strain was tested (along with
the BECh1 and A 1560 strains) for production of secondary metabolites under optimized
conditions. The strain produced only kojic acid at a level of approximately 15% of that produced
by the A1560 and BECh1 strains cultivated under the same conditions. The BECh2 strain was
also repeatedly tested for production of 3-β-nitropropionic acid in several mycotoxin-inducing
media in combination with various cultivation conditions such as temperature and aeration. The
compound was detected only under one combination of the cultivation conditions.
The BECh2 strain was used as a host for the asparaginase gene, which was derived from the A.
oryzae strain A1560. The asparaginase production strain was obtained by transformation of the
BECh2 strain with the expression plasmid pCaHj621. The expression plasmid was stably
integrated into genomic DNA of the host strain. All DNA sequences comprising the expression
plasmid are known. The plasmid carries the origin of replication from the Escherichia coli vector
pUC19 and the asparaginase coding sequence from A. oryzae strain A1560 placed under the
control of regulatory sequences, promoter and terminator. The promoter and terminator
sequences were derived from Aspergillus niger and Aspergillus nidulans. Both the promoter and
terminator sequences are well-characterized and do not contain any coding DNA.
The
expression vector also contains the URA3 gene from baker’s yeast Saccharomyces cerevisiae and
the amdS gene from A. nidulans. Both genes are well-characterized and serve as selectable
markers during the construction of the asparaginase production strain. The expression plasmid
does not contain antibiotic resistance genes or any other coding sequences besides those
discussed above.
The asparaginase production strain, designated as pCaHj621/BECh2#10, is expected to retain the
secondary metabolite profile of the BECh2 host strain, i.e., to be capable of producing reduced
Asparaginase from A. oryzae encoded by the asparaginase gene from A.oryzae (CTA) 2007 - Page 3(7)
levels of kojic acid and trace levels of 3-β-nitropropionic acid under specific cultivation
conditions. However, the synthesis of these secondary metabolites is not expected under
conditions normally used in the large-scale production of enzymes intended for use in food. To
confirm that no kojic and 3-β-nitropropionic acids were formed during asparaginase production at
toxicologically significant levels, one batch of the asparaginase enzyme preparation was analyzed
for these compounds. Neither kojic acid nor 3-β-nitropropionic acid were detected at the
respective detection limits of 1.4 mg/kg and 0.6 mg/kg.
The BECh2 strain was used by Novozymes as a host strain in the construction of production
strains for xylanases from Aspergillus aculeatus and Thermomyces lanuginosus, glucose oxidase
from A. niger, triacylglycerol lipase from T. lanuginosus/Fusarium oxysporum, and
phospholipase A1 from Fusarium venenatum. The DNA introduced into these production strains
was essentially the same as that introduced into the asparaginase production strain, except for the
DNA sequences encoding the individual enzymes.
3.3. Fermentation, recovery, and formulation
Asparaginase is produced by submerged fermentation of the asparaginase production strain using
a fermentation medium composed of food-grade materials. The enzyme is secreted to the
fermentation broth. The fermentation is conducted under controlled conditions and monitored for
microbial contamination. If contamination is detected, the fermentation is terminated. After the
fermentation has been completed, the cell mass is separated from the broth using drum filtration
or centrifugation. The clear broth is then subjected to ultrafiltration and/or evaporation to
concentrate and purify the enzyme. During ultrafiltration, low molecular weight components of
the broth are partially removed. To remove the residual microorganisms and insoluble
components of the fermentation broth, the enzyme concentrate is subjected to pre-germ and germ
filtration. The enzyme is then formulated with glycerol, sodium benzoate and potassium sorbate.
4. Characterization
4.1. Asparaginase
Asparaginase described in the Novozymes dossier catalyses the hydrolysis of L-asparagine to Laspartic acid and ammonia. The enzyme also acts on glutamine but has no activity on other
amino acids and asparagine residues in peptides and proteins. The Chemical Abstract Service
Registry Number (CAS No.) of asparaginase is 9015-68-3. Asparaginase is classified by the
Enzyme Commission of the International Union of Biochemistry and Molecular Biology
(IUBMB, online edition) as follows:
Accepted name:
Other name(s):
Reaction:
Systematic name:
EC:
asparaginase
asparaginase II; L-asparaginase; colaspase; elspar; leunase; crasnitin;
α-asparaginase
L-asparagine + H2O = L-aspartate + NH3
L-asparagine amidohydrolase
3.5.1.1
Asparaginase activity is determined by measuring the rate of hydrolysis of L-asparagine to Laspartic acid and ammonia. Ammonia subsequently reacts with α-ketoglutarate to form Lglutamic acid. The reaction is catalysed by glutamate dehydrogenase in the presence of NADH,
which is oxidized to NAD+ with the concomitant loss of absorbance measured at 340 nm. The
Asparaginase from A. oryzae encoded by the asparaginase gene from A.oryzae (CTA) 2007 - Page 4(7)
asparaginase activity is measured as a rate of NADH consumption under standard conditions
(pH=7; 370C). The activity of asparaginase is expressed in ASNU activity units. One ASNU is
defined as the amount of asparaginase that produces one micromole of ammonia per minute under
standard conditions (pH=7; 370C).
Novozymes assessed the potential allergenicity of asparaginase by comparing its amino acid
sequence to the amino acid sequences of known or putative allergens in the SWALL
(SWISSPROT AND TrEMBLE) and GeneBank databases. It has been reported that an
immunologically significant sequence identity requires a match of at least eight contiguous amino
acids (Metcalf et al., 1996). No amino acid sequence identity between asparaginase and known
or putative allergens was detected at the level of 7 and 8 contiguous amino acids. The amino acid
sequence of asparaginase was also assessed for sequence homology with protein sequences that
contained the word “toxin” in any of the description fields in the SWALL and GeneBank
databases. No significant sequence homology was detected.
Asparaginases are produced by a number of microorganisms, including bacteria and fungi. In
many of these microorganisms, two forms of the enzyme are produced, a cytoplasmic and a
periplasmic or extracellular asparaginase. The A. oryzae asparaginase is an extracellular enzyme.
4.2. Asparaginase enzyme preparation
The asparaginase enzyme preparation is marketed under the name Acrylaway® L. The typical
composition of the asparaginase enzyme preparation is (approximately):
Total organic solids (TOS):
Water:
Glycerol:
Sodium benzoate:
Potassium sorbate:
4%
46%
50%
0.3%
0.1%
TOS is defined as:
TOS (%) = 100 – (A + W + D), where:
A = % ash, W = % water and D = % diluents and/or other formulation ingredients (JECFA,
2006). The typical asparaginase activity of Acrylaway® L is 3500 ASNU/g.
The asparaginase enzyme preparation conforms to the General Specifications for Enzyme
Preparations Used in Food Processing (JECFA, 2006). It does not contain significant levels of
side activities because the genes encoding extracellular amylases and proteases were deleted from
the genome of the asparaginase production strain. The enzyme preparation does not contain
viable cells of the production organism.
5. Functional uses
Asparaginase is intended for use as a processing aid during food manufacture to convert
asparagine to aspartic acid in order to reduce the formation of acrylamide. Acrylamide is formed
from asparagine and reducing sugars primarily in starchy foods that are baked or fried at
temperatures above 1200C. The reaction between sugars and asparagine proceeds through several
Asparaginase from A. oryzae encoded by the asparaginase gene from A.oryzae (CTA) 2007 - Page 5(7)
intermediates with acrylamide as the final product formed from the side chain of asparagine. The
formation of acrylamide may be reduced by removal of asparagine via enzymatic conversion with
asparaginase to aspartic acid. Asparaginase will be primarily used during production of starchy
foods that are major sources of dietary acrylamide, such as potato chips, French fries, tortilla
chips, and dough-based products, including cookies, crackers, pretzels, crisp bread, and biscuits.
Novozymes assessed acrylamide reduction in several foods using different levels of asparaginase.
Acrylamide reduction ranged from 40 to 98% depending on the food, asparaginase use level,
temperature, and holding time.
Asparaginase will be used in dough-based products at levels from 200 to 2500 ASNU per 1 kg of
processed food, corresponding to 6-70 g of the asparaginase enzyme preparation (Acrylaway®)
per 100 kg of processed food. For production of French fries and potato chips, potato strips or
slices will be dipped in an asparaginase bath containing approximately 12,000 ASNU/L of water
and held for a specified time. Novozymes estimates that the enzyme dosage per 1 kg of the final
food product would be 1420 ASNU for French fries and 1820 ASNU for potato chips. These
figures are based on the assumption that the maximum water pick-up by potato strips and slices
would be 5% and that 1 kg of raw potato strips or slices would yield 0.42 kg of French fries or
0.33 kg of potato chips.
6. Reactions and fate in food
Asparaginase will be used during food processing prior to heat treatment. The enzyme would be
subsequently inactivated during heat processing, such as cooking, baking, or frying. As a result
of the catalytic activity of asparaginase, low levels of aspartic acid and ammonia are expected to
be formed in food. Asparaginase can also hydrolyze free glutamine with the formation of
glutamic acid.
7. References
Barbesgaard, P., Heldt-Hansen, H.P., and Diderichsen, B., 1992. On the safety of Aspergillus
oryzae: a review. Appl. Microbiol. Biotechnol. 36, 569-572.
Blumenthal, C.Z., 2004. Production of toxic metabolites in Aspergillus niger, Aspergillus oryzae,
and Trichoderma reesei: justification of mycotoxin testing in food grade enzyme preparations
derived from the three fungi. Regul. Toxicol. Pharmacol. 39, 214-228.
IUBMB, online edition: http://www.chem.qmul.ac.uk/iubmb/enzyme/search.html
JECFA, 2005. Phospholipase A1 from Fusarium venenatum expressed in Aspergillus oryzae.
New specifications prepared at the 65th meeting (Geneva, Switzerland, 7-16 June 2005).
Combined
Compendium
of
Food
Additive
Specifications.
Online
edition:
http://www.fao.org/ag/agn/jecfa-additives/search.html?lang=en
JECFA, 2006. Joint FAO/WHO Expert Committee on Food Additives. “General Specifications
and Considerations for Enzyme Preparations Used in Food Processing.” Prepared at the 67th
meeting (Rome, Italy, 20-29 June 2006). FAO JECFA Monographs 1, Vol. 4. Online edition:
http://www.fao.org/ag/agn/jecfa-additives/search.html?lang=en
Asparaginase from A. oryzae encoded by the asparaginase gene from A.oryzae (CTA) 2007 - Page 6(7)
Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L., and Fuchs, R.L.,
1996. Assessment of the allergenic potential of foods derived from genetically engineered crop
plants. Critical Reviews in Food Science and Nutrition 36(S), S165-S286.
Nielsen, P.H., Malmos, H., Damhus, T., Diderichsen, B., Nielsen, H.K., Simonsen, M., Schiff,
H.E., Oestergaard, A., Olsen, H.S., Eigtved, P., Nielsen, T.K., 1994. Enzyme applications
(industrial). In: Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 9, 4th ed. John Wiley
& Sons, New York, pp. 567-620.
Novozymes, 2006. Novozymes A/S. An Asparaginase enzyme preparation produced by a strain
of Aspergillus oryzae expressing the Aspergillus oryzae asparaginase gene. November 9, 2006. A
dossier submitted to JECFA.
Olempska-Beer, Z.S., Merker, R.I., Ditto, M.D., DiNovi, M.J., 2006. Food-processing enzymes
from recombinant microorganisms – a review. Regul. Toxicol. Pharmacol. 45, 144-158.
Tominaga, M., Lee, Y.-H., Hayashi, R., Suzuki, Y., Yamada, O., Sakamoto, K., Gotoh, K., Akita,
O., 2006. Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus
oryzae RIB strains. Appl. Environ. Microbiol. 72, 484-490.
Van den Broek, P., Pittet, A., and Hajjaj, H. 2001. Aflatoxin genes and the aflatoxigenic potential
of Koji moulds. Appl. Microbiol. Biotechnol. 57, 192-199.
Watson, A.J., Fuller, L.J., Jeenes, D.J., and Archer, D.B., 1999. Homologs of aflatoxin
biosynthesis genes and sequence of aflR in Aspergillus oryzae and Aspergillus sojae. Appl.
Environ. Microbiol. 65, 307-310.
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